PhoP Regulatory Domain

=Introduction= The response regulator PhoP from the OmpR/PhoB family of two-component systems is responsible for initiating the cellular response to the extracellular concentration of Mg2+ of certain gram-negative bacteria such as Escherichia coli and Salmonella enterica. A drop in Mg2+ concentration is an indicator to the pathogenic bacteria that it has entered a host cell and needs to react correspondingly. The histidine protein kinase, PhoQ, spanning the inner membrane of the bacteria, senses the lowered Mg2+ concentration and phosphorylates the cytosolic PhoP. PhoP forms a homodimer and sets a signal cascade in motion, affecting other two component signalling systems and directly regulating gene expression by binding to PhoP boxes on the DNA. PhoP consists of a regulatory domain and an effector domain.

Gene regulation is achieved by the increased affinity of the homodimer to the PhoP box, a tandem repeat promoter. The response is organism specific but generally involves virulence and the survival in an environment with low Mg2+ concentration.

=Structure= PhoP consists of 2 domains, the regulatory domain and the C-terminal effector domain.

Regulatory Domain
The regulatory domain consists of 5 α-helixes and 5 β-sheets. Twofold symmetry is achieved on the α-4 helix, β-5 sheet and α-5 helix face. The regulatory domain may be phosphorylated at a conserved aspartate by PhoQ, a histidine protein kinase. Phosphorylation of this aspartate stabilizes the homodimer. The PhoP regulatory domain has intrinsic autophosphatase activity, allowing it to inactivate itself after a delay.

Activated form

 * Phosphorylation of the regulatory domain stabilizes dimer formation.
 * The phosphoryl analog Beryllofluoride (BeF3-) was used during crystalization of the regulatory domain of Escherichia coli.
 * The Phosphoryl analog made the following bonds (Fluoride: light green, Magnesium: dark green):
 * BeF3-
 * F1 to Mg2+
 * F2 to Thr 79 (Hydrogen Bond)
 * F3 to Lys 101 (Salt bridge)

Un-activated form

 * Under normal physiological conditions, unactivated PhoP occurs mainly as a monomer. At higher concentration unactivated PhoP has been shown to dimerize and act similar to activated and dimerized PhoP. Many regulatory domains isolated from members of the OmpR/PhoB family and in their inactive form, crystalize in a form similar to their activated dimers.

Effector domain
When the regulatory domain is phosphorylated, PhoP forms a homodimer. There is no direct change in conformation conferred on the effector domain by the regulatory domain. It is the dimerization that activates the effector domain. The PhoP has a winged helix-turn-helix motif characteristic of the OmpR/PhoB family of response regulators. This winged helix-turn-helix allows binding to DNA and regulation of transcription. Binding occurs at tandem repeat promoters with two repeats of the sequence (T/G)GTTTA, known as the PhoP box.

=Function= Two component systems allow bacteria to respond to changes in their environment. These systems are found mainly in prokaryotes and a few eukaryotes. The PhoP/PhoQ system, found specifically in gram-negative bacteria, react mainly to a drop in extracellular Mg2+. Since the magnesium concentration is typically lower inside the host cell compared to outside, this acts as a trigger to become virulent. Other functions activated by the PhoP/PhoQ system includes adaptation to low Mg2+ conditions, changes in cell wall, expression of proteases to protect against antimicrobial peptides and various other species specific responses. PhoP in Salmonella enterica regulates up to 40 proteins. PhoP is not limited to direct regulation of gene expression. PhoP may regulate other two component systems at transcription, posttranscription and posttranslation too, in fact PhoP activates the expression of its own phoP gene, resulting in positive feedback and definite conversion to virulence. Further evidence to PhoP's role in response to the extracellular environment is given by evidence of sRNA regulation of the expression of the phoP gene. Envelope stress, though σE and the sRNA MicA affects expression of the PhoPQ system.

The PhoP/PhoQ system may also be found in non-cytoplasmic pathogens, such as ''Erwinia carotovora supsb. carotovora'', a plant pathogen living in the intercellular fluid, or non-pathogenic bacteria.

=Importance of PhoP= PhoP/PhoQ plays in key role in certain bacteria becoming virulent. For example, Salmonella typhimurium becomes avirulent when it carries a phoP mutation. This makes it a promising target for vaccine and antimicrobial drug development. =References=